Response of Baboon Peripheral Blood Lymphocytes to Phytohemagglutinin: Time-Course and Dose-Response Relationships

Abstract

Optimal conditions for studying phytohemagglutinin(PHA)-induced transformation of baboon peripheral blood lymphocytes were determined. Maximal stimulation, as determined by uptake and incorporation of tritiated thymidine (total cell and trichloroacetic- acid-precipitable radioactivity), occurred at a PHA level of 12.5 μg in a culture volume of 0.25 ml containing 2 x 10^5 lymphocytes. Optimal stimulation occurred after a total incubation period of 114 h, the last 18 h of which was in the presence of 1 μCi of the labeled DNA precursor per culture. While there was considerable variation in the extent of responsiveness of lymphocytes from individual animals, the shape of the doseresponse and time-course curves for most mitogen concentrations was generally similar.

A micro-culture technique for the mitogen stimulation of lymphocytes fromMacaca mulatta (rhesus) andMacaca fascicularis (cynomolgus)

“…In our study the total culture volume was 0.25 ml which was comparable with that of.2 ml (WILSON et al, 1978;TAYLOR, MARCI-mTrE &SmDIQUI, 1978) and .24-.25 ml (GREEm~, 1977;HUMBErt & HETHERINGTON, 1981). However, the semi,micromethods required a culture volume of 4.06--4.2 ml (JtrNGE et al, 1970) and 5.4 ml (DE VgIES et al, 1975).…”

Blastogenic response of peripheral blood lymphocytes to mitogens in cynomolgus monkeys (Macaca fascicularis)

“…In general, the extent of in vitro lymphocyte blastogenesis is considered to correlate with in vivo cell-mediated immune response, such as delayed hypersensitivity in human and guinea pig systems [Oppenheim, 19681. As to nonhuman primates, the investigations on mitogen-induced blastogenesis have been done with rhesus monkeys [Mackler et al, 1973;Taylor et al, 19781, baboons [Greene et al, 1977;Eichberg et al, 19811, stumptailed monkeys [Reed, 19761, marmosets [Harvey et al, 1974;Kateley et al, 19771, cynomolgus monkeys [Humber & Hetherington, 19811, and so on. Despite the increasing use of cynomolgus monkeys as experimental models for various human disease, few basic studies concerning their cell-mediated immune response have been undertaken.…”

Effects of NO2on the response of baboon alveolar macrophages to migration inhibitory factor

“…As additional confirmation that the baboons' lymphocytes were capable of producing and releasing MIF, the cells were stimulated with the nonspecific mitogen PHA, an agent known to induce blastogenesis in baboon lymphocytes (Greene et al, 1977). It also has been shown to stimulate the production and/or release of MIF from human lymphocytes (Papageorgiou et al, 1974;Lomnitzer et al, 1975).…”

“…After centrifugation (265 Xg, 30 min, 22°C) the cells at the interface were removed with a sterile Pasteur pipette and washed three times with PBS by centrifugaron (385 Xg, 10 min, 22°C). The washed cells were resuspended in RPMI 1640 (Grand Island Biological Co., Grand Island, N.Y.) containing 25 m/ W HEPES buffer, 2 mg/ml sodium bicarbonate, 10% heat-inactivated fetal calf serum, and supplemented with 200 m/ W L-glutamine and penicillin and streptomycin, hereafter referred to as a complete medium, following the method of Greene et al (1977) as modified from Thurman et al (1973). A sample of cells was removed for viability assessment as determined by the ability to exclude 0.2% Erythrocin B, and for differential counting using Turks solution.…”

“…Mitogenic Stimulation of Lymphocytes with Phytohemagglutinin (PHA) The ability of peripheral lymphocytes from normal and NO 2 -exposed animals to respond to mitogenic stimulation with PHA was determined by using procedures that we have found to be optimal for baboon lymphocytes (Greene et al, 1977). Lymphocytes were adjusted to a concentration of 10 6 /ml in medium RPMI 1640 containing 10% fetal calf serum and supplemented as above, and dispensed in 0.2-ml portions containing 2 X 10 s cells into flat-bottomed microtissue culture plates (Linbro, Hamden, Conn.).…”